Germicidal compositions



assmss Patented June 6, 1961 2,987,435 GERMICIDAL COMPOSITIONS ThomasGwyn Davies, Vaucluse, New South Wales, Australia, and Doreen LoisWedderburn, London, England, assignors to Lever Brothers Company, NewYork, N.Y., a corporation of Maine No Drawing. Filed Aug. 6, 1958, Ser.No. "753,419 Claims priority, application Great Britain Aug. 8, 1957 9Claims. (Cl. 16718) This invention relates to germicidal compositionswhich are also bleaching agents.

Quaternary ammonium salts are widely used for their germicidalproperties and are especially valuable not only because they show highgermicidal activity against both gram positive and gram negativeorganisms in dilute solution, but also because of their stability, lackof odor and lack of toxicity. When a substrate material is treated witha solution of a quaternary salt and the material is then washed withwater, the. material generally retains a proportion of the germicidalactivity of-the quaternary salt against gram positive organisms, butretains very little, if any, activity against gram negative organisms:the gram positive germicidal properties can thus be said to besubstantive to the substrate material, while the gram negativegermicidal properties are not substantive.

It has now been discovered that when a quaternary ammonium salt isincorporated in a hypochlorite solution that the substantive grampositive germicidal proper-v ties are enhanced synergistically and theformerly nonsubstantive gram negative germicidal properties becomesubstantive and also are synergistically enhanced. Substantive is usedin the normal sense to mean that the quaternary ammonium compounds andtheir germicidal properties are substantially permanently attached tothe material. It has been found that when proteinaceous and cellulosicmaterials are treated with a quaternary ammonium salt in conjunctionwith an aqueous sodium hypochlorite solution, for example, they retainafter washing synergistically better germicidal properties than when aquaternary ammonium salt is used alone. Moreover, the sodiumhypochlorite retains its bleaching properties.

The compositions of the invention accordingly comprise a hypochloritesolution in which is incorporated a germicidal qua-ternary ammoniumsalt.

The hypochlorite solution will normally be aqueous sodium hypochloritesolution, though other hypochlorites, for instance potassium and calciumhypochlorites' or mixtures thereof, can also be used. Commercialhypochlorite bleaching solutionsare suitable.

wQuaternary ammonium salts which are suitable are those which have asubstantial germicidal activity, for example benzalkonium chloride(which is a mixture of alkyldimethylbenzylammonium chlorides in whichthe allgyls range from C l-1 to C H cetyl pyridinium bromide, cetyltrimethylammonium chloride and fi-(p-tertoctylphenoxyethyl) diethylbenzylammonium chloride. Other substances which can be used are laurylpyridinium chloride and dodecylbenzyltrimethylammonium chloride. Thecompositions of the invention are conveniently obtained by adding thequaternary ammonium salt, as such or in aqueous solution, to an aqueoushypochlorite solution, and mixing the components together. Preferablythe materialsadde'd are such that the composition obtained' contains theequivalent of at least 0.1% by weight of available chlorine and 0.01%quaternaryammonium salt. Very suitable are composi-' tions based onsodium hypochlorite solution containing from :l to by weight ofavailable chlorine. Preferably from 0.05 lto 20 parts of quaternaryammonium salt are incorporated for each part by weight of chlorine, Itis'convenient to prepare a composition which is relatively by weight ofconcentrated, and which can be diluted with water before use. An aqueoussodium hypochlorite solution containing 0.25 to 1.5%, for instance 0.5or 1%, by weight of available chlorine to which is added a suitableamount of quaternary ammonium salt, for instance 5% (or 2.5 to 7.5%) ofthe salt by weight of the solution is very suitable for use as agermicidal bleaching agent after 40 told dilution. The relativeproportions of hypochlorite and quaternary ammonium salt which aresuitable in any given instance will depend upon the particular use towhich the composition is to be put, and accordingly upon the extent ofbleaching and germicidal properties which are desired.

When a quaternary ammonium salt is added to a relatively concentratedhypochlorite solution, there is usually some form of phase separation: acloudy liquid or emulsion may be obtained which on standing may separateinto two layers; alternatively, a crystalline precipitate may be throwndown after standing for some time. This may be due to complex formationbetween the ingredients of the composition. The degree of inhomogeneitywill depend not only on the concentrations and the nature of theingredients used, but also on the age of the composition. Unwantedinhomogeneity can be avoided by using suitable concentrations of theingredients, or in some cases by adding dispersing agents.

The invention also includes a process in which materials are treated bycontacting them with a composition of the invention. The process isparticularly suitable for use with keratinous fibers, for instance humanhaid and wool, and with cellulosic fibers. It has further beendiscovered that the synergistically enhanced substantive germicidalproperties can be given to the material by bringing the two componentsof the compositions of the invention (that is, the hypochlorite and thequaternary ammonium salt) together on the material itself. Thus thehypochlorite and the quaternary ammonium salt can be used successivelyto treat the material to which the properties are to be given. Forinstance the material can betreated first wit han aqueous hypochloritesolution containing 0.1% (or 0.5% or 1%) available chlorine and thenwith a 0.02% aqueous benzalkonium chloride solu tion. Such processes arealso within the scope of the invention.

The invention is illustrated by the following examples.

EXAMPLE 1 'To 10 g. of an aqueous sodium hypochlorite solutioncontaining 5% by weight of available chlorine was addedby weight aqueoussolution of benz' alkonium chloride (a benzyl'dimethyl alkylammonium '1g. of a 50% chloride in which the alkyl group is a long'chain alkylgroup having an The composition obtained on shaking the two componentstogether was a cloudy liquid containing 4.5% by weight of availablechlorine and 4.5% by weight of benzalkom'um chloride, representing acomposition according to the invention.

The synergistically enhanced substantive germicidal properties of thecomposition towards cotton poplin fabric was tested as follows. A stripof a cotton poplin (0.5 g.)

' was immersed in the composition 100 cc.) for 5 minutes,

containing 5% available-chlorine and washed and dried. in the same way,

removed and washed 3 times with distilled water cc.), and dried. Three 1cm. squares were cut from the' strip and placed one each on the surfaceof three agar plates seeded respectively with the organisms Ps.fluorescens, Staph. albus andStreptococcus bovis.

(The first of these organisms is gram negative and the others are grampositive.) Simultaneously squares of poplin treated by immersion inaqueous sodium hypochlorite solution and also squares similarly treatedwith average length of 13 carbon atoms);

Zone of inhibition (mm.)

Treatment of poplin 1 Ps. fluo- Staph. Strep.

T8SC6TLS albus bovis Hypochlorite "l -i i 1 0.5 Ben'zalkonium chloride 06 3 Hypochlorite and benzalko um c oride 6 11 8 The synergisticallyenhanced substantive germicidal properties of the composition containinghypochlorite and benzalkonium chloride are clearly shown.

The composition was further tested for its activity on dilution bytreatment of cotton poplin in the same way as described above, but with'5 fold, 10 fold, 20 fold and 40 fold dilutions of the composition withdistilled water, using the organisms Ps. fluorescens and Staph. albus.Similar tests were carried out with corresponding dilutions ofbenzalkonium chloride and sodium hypochlorite separately. The zone testsin each instance showed a synergistic effect between the two componentsin the composition, giving synergistic residual germicidal activityagainst the two organisms.

EXAMBLE 2 Test A The synergistically enhanced substantive germicidalproperties of this composition towards cotton poplin was tested usingthe procedure of Example 1. The results were as follows:

Distance of inhibition (mm.)

Treatment of poplin r Ps. fluo- Staph. Strep. Tescens albux bovzsHypochlorite 1 o. t 0 Benzalkonium chloride 0 6 3 Hypochlorite andbenzalkoninrn chloride 6 10 9 Test B The synergi'stically enhancedsubstantive germicidal properties of the composition towards hair wasnext tested by the following procedure. A l g. sampleof human hair wasimmersed for minutes in 100 cc. of a solution prepared by diluting 10cc. of the composition with water (that is, at 10 fold dilution); Thehair was then removed, washed three times 'with distilled .water anddried. Different quantities of the treated hair (1, 25, 50, 100 and 150mg.) were 'theuWeighed out and placed in bottles containing 20 cc. ofRogosa broth which had been inoculated with '1 cc. of aturbid culture ofL'acto bacillus casei, a gram positive lactic acid producing organism;the broth containinghair wasjin'cubated at 37 C. for 24'hour1sand thepH'or" the broth then measured. i

Further samples of hair were prepared as described above but usingdifierent treating solutions, namely a solution prepared by diluting 20cc. of the composition to cc. with distilled water (that is, at 5 folddilution); solutions prepared by diluting 10 cc. and 20 cc. respectivelyof an aqueous solution of sodium hypochlorite containing 5% by weight ofavailable chlorine to 100 cc., and solutions prepared by diluting 10 cc.and 20 cc. respectively of an aqueous solution containing 0.5 by weightof benzalkonium' chloride to 100. cc". These samples were added inweighed amounts to tubes of Rogosa'broth, incubated and the pH of the,broth measured after 24 hours as before. Control experiments in which nohair was placed in the inoculated broth or in which the inoculated brothwas refrigerated were also carried out. These were accordingly upper andlower limit controls. The results were as follows:

pH of incubated broth according to-amount of hair added Treatment ofhair (mg) (At 10 fold dilution):

Hypochlorite and benzalkonium chloride 5.15 5. 30 5. 35 5. 40 5. 50Hypochlorite alone 5. 20 5. 20 Benzalkonium chloride alone 5; 15 5'. 205. 25 5. 25 5. 25 (At 5fo1d dilution):

Hypochlorite and benzalkonium chl0ride 5.15 5. 32 6. 47 5. 55 5.65Hypochlorite alone 5. 20 Benzalkonium chloride a1 5. 25 5. 25 5.25 5. 30

The incubated control broth had a pH of 5.07, while the refrigeratedcontrol had a pH of 5.80. p

The extent of reduction of acidity, or increase of, pH, in the brothcontaining treated hair is a measure of the reduction in activity, andhence in, growth, of the Lactobacillus, and is thus a measure of thegermicidal properties retained by the treated hair. The superior resultsobtained with treatment of hair using the composition of the inventionas compared with its separate ingredients are clearly shown.

' Test C In another test, the synergistically enhanced substantivegermicidal activity of the composition towards cotton poplin fabric wascompared with that of benzalkonium chloride alone using the followingprocedure. Cotton poplin was immersed for 5 minutes in'the compositioncontaining 5% of available chlorine and 05% of benzalkonium chloridefurther diluted 10 fold with distilled water; then removed, washed wellwith distilled water and dried.- -Another sample of cotton poplin wassimilarly treated except that it was immersed in 0.5% benzalkoniumchloride solution diluted 10 told without any hypochlorite. The twocopies of treated poplin were cut up and weighed out into two sets ofbottles containing 20 cc. of nutrient broth, in varying amounts (1, 5,'10 and 50 mg. respec tively). A bottle containing broth with no addedpoplin and one with unseeded broth were prepared as controls.

The bottles were each seeded with 0.1 odor-Staph. albus and incubated at37 C. for 24 hours. "The turbidity ofeach broth sample was thendetermined using a nephelorneter, and gave an estimate of the relativenumbers of bacteria present and hence of the germicidal activityretainedby the treated fabric. The results were as follows.

EXAMPLE 3 ried out using 10 fold and 100 fold dilutions with distilledwater for treatment of the poplin. Similar tests were conducted using 5%aqueous benzalkonium chloride solution without any hypochlon'te. Thezones of inhibition were measured as in Example 1. I

Organism Staph. albus I Ps. fluorescem Dilutlon.

Nil I 10 fold I100 fold Nil Distance of inhibition (mm) Compositionused:

Quaternary (5%) with no hypochlorite 9 8 5. 5

Quaternary 4.5% 5116 E551 ehlorite:

It Will be seen that even when as little as one part by weight ofavailable chlorine was present to ten parts by EXAMPLE 4 This exampleillustrates four compositions of the inven 'on containing difl erentquaternary ammonium salts, and compares their synergistically enhancedsubstantive germicidal activity with that of each of their ingredients,against four different organisms. .The quaternary ammonium saltsemployed were (A) benzalkonium chloride, (B) 13-(p-tert.octylphenoxyethyl) diethyl benzylammonium chloride, (C) cetylpyridinium bromide, and (D) cetyl trimethylammonium chloride. Thecompositions were obtained by adding 4% by weight of quaternary ammoniumsalt to a sodium hypochlorite solution containing by weight of availablechlorine: a cloudy solution or emulsion was obtained in each case,except with cetyl pyridinium bromide, which gave a clear yellowsolution.

Cotton poplin squares were treated using the procedure of Example 1 andemploying the dilferent compositions; agar plates seeded with theorganisms Staph. albus, Strep. bovis, B. c011 and Ps. fluorescens wereused (the first two of these are gram positive and the last two gramnegative). Squares of poplin were treated with a sodium hypochloritesolution containing 5% by weight of available chlorine, with each of thecompositions, and with aqueous 4% by weight solutions of each of thequaternary ammonium salts alone. The results were as follows.

Distance of inhibition (mm.) Treatment Staph. Strep. 8. e011 Ps.flualbus bouts orescem Hypoehlorite alone 1 1 1 1 Quaternary salt (A)with hypoehlo- 13 7 2 8 rite alone. 6 3 0 0 Quaternary salt (B) withhypochlo- 11 6 2 2 rite alone. 1 6 1 0 0 Quaternary salt (0) withhypochlo- 10 8 10 5 rite alone. 5 4 0 0 Quaternary salt (D) withhypochlo- 5 4 3 2 rite alone. 2.5 1 0 0 EXAMPLE 5, p

The synergism between benzalkonium chloride (B.A.C.) and pure 10% sodiumhypochlorite solution and also between'cetyl trimethyl ammonium bromide,Cetavlon, calculated as C H (CH NBr and pure 10% sodium hypochloritesolution were investigated using the following procedure.

A piece of cotton poplin (0.5 g.) was shaken in 100 cc. of test solutionfor two minutes, washed three times with 100 cc. distilled water anddried overnight. Squares (1 cm.) of the treated poplin were then laid onplates interfor 24 hours at 37 C.

Test solutions used contained '(a) quaternary salt, (b) hypochlorite,(c)- quaternary and hypochlorite, and the results are given in thefollowing table.

Zones of Inhibition in Millimeters Pseudo- Staph. Strep. monas B. al usbovis fluorescolt lia e 2 g 9 2. 5 1 0 NaOOl. 2.5 0 0 0 0 L25 15 11 2.52.5 NaOC-l 10 1 0 1 0 getggllom- 12 7 4 0 0 a a. +Oetavlon +5 12 12 1 4aOOL. 5 0 0 0 0 Oetggllonu 2 g 5 4 0 0 +Cetavlon +2.5 10 11 1 4 Na0OI2.5 0 0 0 0 oet ng on 2. 2 6 5 0 0 a +Oetavlon +1.25 8 10 1 2 Theresults show conclusively a synergistic effect. This fibrous materialsas well as to bleach said materials. The

1. A process of cleaning and disinfecting a material which comprises thesteps of contacting said material with 7 a solution of sodiumhypochlorite containing from 0.45% to about 5% of available chlorine andthereafter contacting said material with an aqueous solution containingfrom about 0.05% to about 5.0% by weight of benzalkonium chloride.

2. The process of cleaning and disinfecting a material wherein thematerial is contacted with a germicidal composition comprising sodiumhypochlorite and benzalkonium chloride, the sodium hypochlorite beingpresent in an amount suflicient to provide from about 0.45% to about5.0% by weight of available chlorine, and the benz alkoniurn chloridebeing present in an amount ranging from about 0.5%, to about 5.0% byweight.

3. A process as set forth in claim 2, wherein the material is akeratinous fiber.

4. A process as set forth in claim 2, wherein'the material is acellulosic'fiber.

5. A germicidal composition comprising sodium hypochlorite andbenzalkonium chloride, the sodium hypochlorite providing from about0.45% to about 5 .0% by weight of available chlorine, and saidbenzalkonium chloride being present in an amount ranging from about 0.5%to about 5.0% byweight.

6. A germicidal composition comprising sodium hypochl-orite and cetyltrimethyl ammonium bromide wherein the weight ratio of the cetyltrimethyl ammonium bromide to the available chlorine from the sodiumhypochlorite is approximately 1:1.

7. -A gernucidal composition comprisin'g'sodium hypo 8 chlorite Iand'18-(p tertoctylphenoxyethyl) diethyl benzylammonium chloride'whereinthe weight ratio of the henzylammonium chloride wherein theweightratiopf the benzyl'ammonium chlorideto the availablechlorine fromthe sodium hypochloriteisapproximately 4:5.

8. A germicidal composition comprising sodium hypochlorite and cetylpyridinium bnomide wherein the weight ratio of the cetyl pyridiniumbromide to the available chlorine from the sodium hypochlorite isapproximately 4:5.

9. A germicidal composition comprising sodium hypochlorite and cetyltrimethyl ammonium chloride wherein the weight ratio of the cetyltrimethyl ammonium chlorride to the available chlorine from thehypochlorite is approximately 4:5.

References Cited in the file of thispatent UNITED STATES PATENTS[Halvorson Nov. 25, 1941 Kalusdian May 25, 1943 OTHER REFERENCES SoapJournal, September 1956, vol. 22, p. 58.

UNiTED STATQS PATENT OFFICE oE'rEieM or Patent No. 2,987,435 June 6,

Thomas Gwyn Davies etall certified that error appears in the abovenumbered pat- It is hereby the said Letters Patent, should read as entrequiring correction and that corrected below Column 2 line 30, forhaid" read hair line 40 for "wit han" read with an column 8, lines 3 and4 strike out "wherein the weight ratio of the benzylammonium choride".

(SEAL) Attest:

DAVID L. LADD Commissioner of Patents ERNEST W. SWIDER Attesting OfficerUSCOMM-DC" UN iTED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PatentNo. 2,987,435 June 6 1961 i Thomas Gwyn Davies et a1 error appears inthe above numbered pat- It is hereby certified Chat t'bers Patent.should read as ent requiring correction and that the said Le "correctedbelow.

line 30, for "haid" read hair line 40,

column 8, lines 3 and 4,

of the benzylammonium Column 2 for "wit han" read with an strike out"wherein the weight ratio choride".

(SEAL) Attest:

ERNEST W. SWIDER Attesting Officer DAVID L. LADD Commissioner of PatentsUSCOMM-DC-

1. A PROCESS OF CLEANING AND DISINFECTING A MATERIAL WHICH COMPRISES THESTEPS OF CONTACTING SAID MATERIAL WITH A SOLUTION OF SODIUM HYPOCHLORITECONTAINING FROM 0.45% TO ABOUT 5% OF AVAILABLE CHLORINE AND THEREAFTERCONTACTING SAID MATERIAL WITH AN AQUEOUS SOLUTION CONTAINING FROM ABOUT0.05% TO ABOUT 5.0% BY WEIGHT OF BENZALKONIUM CHLORIDE.